I appreciate the editorial feedback provided by Holly Rahurahu and Sam Shaffer.

The experiments and protocols have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of North Carolina at Chapel Hill (mice) and Fairfield University (rats) and follow the guidelines of NIH10.
1. Restraint stress in mice
NOTE: Mice offer a number of advantages when studying the effects of stress on physiology and neuronal systems. With the prevalence of transgenic mouse lines, mechanistic investigations into the interactions between genes and stress exposures are readily feasible. Whether using transgenic or wild-type mice, consideration of the background strain is recommended. Additionally, due to their size and social housing, mice offer an advantage in space utilization over other species.
<ol>
	<li>Restraint stress exposure
	<ol>
		<li>Randomly assign mice to homecage control, acute restraint stress, or chronic restraint stress groups.</li>
		<li>Determine the length of stress exposure. For acute stressors, use a single session lasting 5 min up to 12 h confined in the restraint apparatus. For chronic stressors, ensure that the session matches the length of the acute stressor but is repeated daily for as short as 3 days and as long as 21 days. 
		NOTE: Because mice will be without access to food and water while in the restraint conditions, consider whether homecage control animals should also lack access to food and water.</li>
		<li>Modify 50 mL conical tubes to add ventilation holes.
		<ol>
			<li>Use a power drill with a 1/8&#34; bit to evenly space holes around each conical tube to ensure airflow throughout all areas of the tube.</li>
			<li>Place these holes to allow the mouse access to air regardless of which direction the mouse is facing in the tube. Ensure no sharp edges remain after modification. 
			NOTE: These modified conical tubes should be effective for most adult mice (~20-40 g) but may not restrain small/young mice or larger strains.</li>
		</ol>
		</li>
		<li>In a testing room, separate from animal housing and behavioral testing, place the restraint-assigned animal into the modified conical tube with the cap attached to confine the mouse in the tube for the designated length of restraint. 
		NOTE: The specifics of the testing room design can vary based on the space available in the facility; the room could include fume hoods and other laboratory equipment and/or laboratory tables/benches or be an empty procedure room. However, regardless of the room setup, the restraint room environment should remain consistent over repeated restraint exposures to avoid context-induced alterations in stress responses11.</li>
		<li>Place the restraint tube horizontally on a flat surface. If needed, secure the tube with laboratory tape or a pipette basin/reagent reservoir so it does not roll with the mouse inside. 
		NOTE: Under stress conditions, mice produce ultrasonic vocalizations. Consider whether the study design requires sound isolation from other restrained subjects12. Corticosterone and other markers of stress responses follow circadian rhythms. As such, considerations regarding the lighting conditions and time of stressor should be made. Across stress groups, restraint should occur at a consistent time of day, preferably early in the light cycle13,14,15. For the study presented here, mice were restrained for approximately 3 h following lights on.</li>
		<li>Monitor each animal during restraint stress at least every 20 min. Visually determine whether the animal is displaying unusual responses (slow breathing, lack of movement). If observed, remove the mouse from the restraint tube, contact a veterinarian, and exclude the mouse from the study.</li>
		<li>Return the mouse to the home cage and provide access to food and water.</li>
	</ol>
	</li>
</ol>
2. Restraint stress in rats
NOTE: As larger rodents, rats have advantages to consider when investigating the effects of stress. Like mice, rats have various strains with differential responses to stress exposure16,17. Additionally, the increased size of rats makes rats easier to use with certain behavioral measures, e.g., drug self-administration.
<ol>
	<li>Restraint stress exposure
	<ol>
		<li>Randomly assign the rat to homecage control, acute restraint stress, or chronic restraint stress groups.</li>
		<li>Determine the length of stress exposure. For acute stressors, use a single session lasting 5 min up to 12 h confined in the restraint apparatus. For chronic stressors, ensure that the session matches the length of the acute stressor but is repeated daily for as short as 3 days and as long as 21 days. 
		NOTE: Because rats will be without access to food and water while in the restraint condition, consider whether homecage control animals should also lack access to food and water.</li>
		<li>For a commercially available restraint tube, follow the steps described below. 
		NOTE: Commercially available restraint tubes for rats are available. These devices have a variety of designs, including metal wire, containers, and plastic/plexiglass tubes with rounded and flat-bottom options. Each option is simple to use.
		<ol>
			<li>Insert the rat into the device, then add a plug adjusted to the subject&#39;s size, and lock it in place. When placed in the restraint tube, ensure that the rat is snugly confined to prevent head-to-tail turns but can breathe easily. 
			NOTE: These devices are designed for restraining rats and do not need modification for respiration. This study uses the tailveiner restrainer (Table of Materials), which includes suction cups on a platform under the tube to secure the device. An additional benefit of this device is that the tail is accessible outside of the device for control of the subject during insertion and removal from the tube or tail vein blood draw during restraint.</li>
		</ol>
		</li>
		<li>In a testing room separate from animal housing and behavioral testing, place restraint-assigned animas into the restraint tube for the designated length of restraint. Place the restraint tube horizontally on a flat surface. 
		NOTE: Under stress conditions, rats also produce ultrasonic vocalizations. Consider whether the study design requires sound isolation from other restrained subjects.</li>
		<li>Monitor each animal during restraint stress at least every 20 min. Visually determine whether the animal is displaying unusual responses (slow breathing, lack of movement). If observed, remove the rat from the restraint tube, contact a veterinarian, and exclude the rat from the study.</li>
		<li>Return the rat to the home cage and provide access to food and water.</li>
	</ol>
	</li>
</ol>
3. Assessments of stress
<ol>
	<li>Measurement of HPA activation
	<ol>
		<li>If appropriate, following restraint stress exposure or removal from homecage, rapidly decapitate subjects to collect trunk blood into heparinized tubes.</li>
		<li>Isolate plasma via centrifugation and store plasma samples at -80 &#176;C. Analyze blood corticosterone using an enzyme-linked immunosorbent assay (ELISA) kit. 
		NOTE: Although corticosterone is often used as a readout of the severity of stress exposure, especially acute stress, corticosterone levels do not always correlate with stress-induced behavioral changes6. Furthermore, the HPA axis response regulating corticosterone release habituates with repeated exposure to the same stressor18. Alternatively, adrenocorticotrophic hormone (ACTH) may be a more reliable measure of the intensity of an acute stressor4. It remains elevated for approximately an hour following the cessation of stressor exposure, but severe and prolonged (approximately &#62; 2 h) stress may fatigue its production.</li>
	</ol>
	</li>
	<li>Behavioral effects
	<ol>
		<li>If appropriate, following restraint stress exposure or removal from homecage, assess the behavioral performance of the rodent with the appropriate behavioral task. 
		&#8203;NOTE: Further information regarding the effects of restraint on various behavioral models can be found in the discussion section.</li>
	</ol>
	</li>
	<li>Neurobiology
	<ol>
		<li>Alternatively or additionally, assess neurobiological function following restraint stress exposure or control. A wide range of assays can investigate restraint stress effects on cellular, synaptic, and circuitry. Depending on the assay, implant an&#160;in vivo&#160;measurement device or harvest brain tissue to perform the selected analysis. 
		NOTE: The effects of stress on cell-type specific changes in protein synthesis and epigenetic modifications have been thoroughly reviewed by McEwen and colleagues1. These genetic and protein modifications induce plasticity to alter synaptic connections and circuitry. In order to conduct these studies, harvest brain tissue following stress exposure and perform genomic, proteomic, ex vivo physiology, and other analyses. Alternatively, assess neurobiological changes through in vivo measurements such as microdialysis, fast-scan cyclic voltammetry (FSCV), fiber photometry, and others. These approaches record neurochemical release and neuronal activation, both following restraint stress exposure2,19&#160;and now during restraint8,9,20. The toolbox to assess the neurobiological mechanisms underlying the effects of restraint stress is rapidly expanding allowing new insights into the complexities of stress.</li>
	</ol>
	</li>
</ol>

Impact of Restraint Stress on Physiology and Neurobiology in Rodent Models

No conflicts of interest to report.

To investigate the effects of stress on physiological, neurobiological, and behavioral functions in rodent models, conditions that produce stress for rodents must be used. Among these considerations are the conditions that produce stress responses in the subject. Restraint is a validated procedure to produce physiological and behavioral responses in rodents matching stress effects in humans. Using a methodology similar to this protocol, restraint has been used to assess a number of molecu...

Due to the universal experience of stress, investigations into the mechanisms of altered responses following stress exposures have been a consistent area of research for several decades1. These investigations have begun to parse out aspects of stress that may be beneficial to increase adaptability and responsiveness to acute stressors from stress experiences that induce maladaptive alterations of physiological and behavioral functions, often resulting from prolonged exposure to repeated and/or unpredictable stressors. Many of these responses to stressors impact the brain function, the hypothalamic-pituitary-adrenal (HPA) axis, the sympathetic and parasympathetic branches of the autonomic nervous system, and the immune system. Exposure to stressors initiates the increased release of corticotropin-releasing factor (CRF) to increase adrenocorticotropic hormone (ACTH) and glucocorticoid hormones, such as cortisol in primates and many other mammals and corticosterone in rodents2. Additionally, epinephrine and norepinephrine are released throughout the periphery and brain as part of the sympathetic nervous system response3. These chemical signals then interact with a number of physiological systems, such as immune function, metabolism, reproduction, and neuronal reactivity4.
Stressful experiences are associated with a myriad of conditions, including increases in anxiety and depression, alterations in learning and memory, impairments in decision-making and executive function, and susceptibility to substance use disorder, among others. Each of these effects is dependent on both parameters of the stressor and the characteristics of the individual experiencing stress4. The nature of the stressor, as acute or chronic and as controllable or unpredictable, substantially alters the outcomes observed. These aspects of stress appear consistent between human experiences and animal models. Thus, to model stress in animal studies, careful consideration must be used to select parameters of stress exposure that are appropriate for the species, sex, and condition.
Although a number of stressors are available for studies with rodents (including psychosocial stressors like social isolation stress or social defeat, pharmacological stressors like cortisol or yohimbine administration, physical stressors like footshock and restraint, and multimodal stressors that combine or alternate between conditions), the focus of this manuscript will be on the use of restraint stress in mice and rats. Restraint stress might be chosen over other stressors because it is easy to perform, inexpensive, adjustable to various sizes of rodents, scalable to both acute and long-term periods, and rarely results in adverse harm to the subject5. Although this manuscript focuses on the lasting changes induced by restraint stress exposure, some studies use restraint in rodents to examine mechanisms and behaviors that occur during restraint, including active coping behaviors involved in escaping the stressor6,7,8,9. The goal of this manuscript is to provide methodological considerations to assess the impact of restraint stress on mice or rat subjects.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>50 mL General Purpose Screwcap Conical Tube</td>
			<td>Globe Scientific</td>
			<td>6288</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Pipette Basin</td>
			<td>Fisher Scientific</td>
			<td>13-681-500</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA Kit for Corticosterone</td>
			<td>Arbor Assays</td>
			<td>K014-H</td>
			<td></td>
		</tr>
		<tr>
			<td>Tailveiner Restrainer Tube</td>
			<td>Braintree Scientific</td>
			<td>RTV</td>
			<td></td>
		</tr>
	</tbody>
</table>

restraint to induce stress in mice and rats

Across all animal species, exposure to stressful conditions induces stress responses. One method to study the effects of stress using rodent models is the restraint stress procedure. Restraint stress has been used for decades to investigate changes in physiology, genetics, neurobiology, immunology, and other systems impacted by stress. Due to the ease of performing the procedure, low cost, and numerous modifications to scale for the intensity and duration of stress exposure, a vast literature of studies has used restraint stress in mice and rats. As one example, this study presents previously published data showing the impact of restraint stress in transgenic mice on plasma corticosterone levels and optogenetically-induced norepinephrine release. Acute restraint stress increased plasma corticosterone levels, yet this effect was blunted in mice following repeat restraint stress. However, stimulated norepinephrine release in the bed nucleus of the stria terminalis was increased only in the repeat restraint stress group. These data highlight important considerations of restraint parameters on dependent measures. Additional descriptions of restraint stress in rats are also included for comparison. Finally, the influence of the parameters of the restraint (e.g., acute vs. chronic) and characteristics of the animal subjects (strain, sex, age) are discussed.

In a previously published study21, the restraint stress procedures described in this manuscript were used to assess the impact of restraint stress on the regulation of cortisol and norepinephrine, falling under the broad possibilities of changes in neurobiology induced by stress mentioned in protocol 3.3. In this study, we utilized the protocol described above for the restraint of mice. Additional details that are beyond the scope of the restraint focus of this article, e.g., the rationale for bra...

Restraint to Induce Stress in Mice and Rats

This article covers the procedures to induce stress responses using physical restraint stress in mice and rats. Additional considerations that should be observed when selecting and using restraint stress in rodent models are discussed.

Across all animal species, exposure to stressful conditions induces stress responses. One method to study the effects of stress using rodent models is the ...

restraint-to-induce-stress-in-mice-and-rats

Research

JoVE Journal

Neuroscience

630 vistas.  Fairfield University. This article covers the procedures to induce stress responses using physical restraint stress in mice and rats. Additional considerations that should be observed when selecting and using restraint stress in rodent models are discussed.

Video: Restraint to Induce Stress in Mice and Rats

Low-stress Route Learning Using the Lashley III Maze in Mice

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other aversive stimuli to motivate task acquisition. As animals age, sensory modalities deteriorate. For example, many strains of mice develop hearing deficits or cataracts. Changes in the sensory systems required to guide mice during task acquisition present potential confounds in interpreting learning changes in aging animals. Moreover, the use of aversive stimuli to motivate animals to learn tasks is potentially confounding when comparing mice with differential sensitivities to stress. To minimize these types of confounding effects, we have implemented a modified version of the Lashley III maze. This maze relies on route learning, whereby mice learn to navigate a maze via repeated exposure under low stress conditions, e.g. dark phase, no food/water deprivation, until they navigate a path from the start location to a pseudo-home cage with 0 or 1 error(s) on two consecutive trials. We classify this as a low-stress behavior test because it does not rely on aversive stimuli to encourage exploration of the maze and learning of the task. The apparatus consists of a modular start box, a 4-arm maze body, and a goal box. At the end of the goal box is a pseudo-home cage that contains bedding similar to that found in the animal&#x2019;s home cage and is specific to each animal for the duration of maze testing. It has been demonstrated previously that this pseudo-home cage provides sufficient reward to motivate mice to learn to navigate the maze1. Here, we present the visualization of the Lashley III maze procedure in the context of evaluating age-related differences in learning and memory in mice along with a comparison of learning behavior in two different background strains of mice. We hope that other investigators interested in evaluating the effects of aging or stress vulnerability in mice will consider this maze an attractive alternative to behavioral tests that involve more stressful learning tasks and/or visual cues.

The Lashley III maze is a route-learning task that does not rely on aversive stimuli or visual cues. It is thus a highly attractive option for evaluating learning and memory, especially in aging mice or otherwise where stress is a consideration.

De baja tensión Ruta de Aprendizaje Usando el laberinto Lashley III en ratones

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other ...

Investigación

Neurociencias

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

El grano y Pruebas de Manejo de Capellini: Simple medidas cuantitativas de la función de las patas delanteras diestro en ratas y ratones

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Un método simple y barato para la Determinación de la sensibilidad al frío y Adaptación en ratones

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood flow that results in a net influx of blood to key organs and an increase in core temperature. This stress-induced hyperthermia is used to assess stress. However, measuring core temperature is invasive. As blood flow is redirected to the core, the periphery of the body can cool. This paper describes a protocol where peripheral body temperature is measured non-invasively in wild blue tits (Cyanistes caeruleus) using infrared thermography. In the field we created a set-up bringing the birds to an ideal position in front of the camera by using a baited box. The camera takes a short thermal video recording of the undisturbed bird before applying a mild stressor (closing the box and therefore capturing the bird), and the bird&#8217;s response to being trapped is recorded. The bare skin of the eye-region is the warmest area in the image. This allows an automated extraction of the maximum eye-region temperature from each image frame, followed by further steps of manual data filtering removing the most common sources of errors (motion blur, blinking). This protocol provides a time series of eye-region temperature with a fine temporal resolution that allows us to study the dynamics of the stress response non-invasively. Further work needs to demonstrate the usefulness of the method to assess stress, for instance to investigate whether eye-region temperature response is proportional to the strength of the stressor. If this can be confirmed, it will provide a valuable alternative method of stress assessment in animals and will be useful to a wide range of researchers from ecologists, conservation biologists, physiologists to animal welfare researchers.

There is a need for a non-invasive assessment of stress. This paper describes a simple protocol using thermal imaging to detect a significant response in eye-region temperature in wild blue tits to a mild acute stressor.

Imágenes Térmicas para estudiar el estrés de forma no invasiva en ilimitados Birds

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood ...

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

Intracerebroventricular Injection of Amyloid-&amp;#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

La inyección intracerebroventricular de péptidos beta-amiloide en ratones normales para inducir de forma aguda déficits cognitivos como la enfermedad de Alzheimer,

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

Un enfoque Simple para inducir Neuritis Autoinmune Experimental en ratones C57BL/6 para las evaluaciones funcionales y Neuropathological

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Utilizando tomografía de coherencia óptica y respuesta Optokinetic como lecturas de sistema Visual estructural y funcional en ratones y ratas

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

A Protocol for Transcranial Photobiomodulation Therapy in Mice

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide range of neurological and psychiatric disorders, and memory enhancement in age-related cognitive decline and neurodegenerative diseases. We describe a laboratory protocol for transcranial photobiomodulation therapy (PBMT) in mice. Aged BALB/c mice (18 months old) are treated with a 660 nm laser transcranially, once daily for 2 weeks. Laser transmittance data shows that approximately 1% of the incident red light on the scalp reaches a 1 mm depth from the cortical surface, penetrating the dorsal hippocampus. Treatment outcomes are assessed by two methods: a Barnes maze test, which is a hippocampus-dependent spatial learning and memory task evaluation, and measuring hippocampal ATP levels, which is used as a bioenergetics index. The results from the Barnes task show an enhancement of the spatial memory in laser-treated aged mice when compared with age-matched controls. Biochemical analysis after laser treatment indicates increased hippocampal ATP levels. We postulate that the enhancement of memory performance is potentially due to an improvement in hippocampal energy metabolism induced by the red laser treatment. The observations in mice could be extended to other animal models since this protocol could potentially be adapted to other species frequently used in translational neuroscience, such as rabbit, cat, dog, or monkey. Transcranial photobiomodulation is a safe and cost-effective modality which may be a promising therapeutic approach in age-related cognitive impairment.

Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

Un protocolo de Transcranial Photobiomodulation terapia en ratones

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide ...

A Revised Surgical Approach to Induce Endolymphatic Hydrops in the Guinea Pig

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) remain unclear. In order to adequately study the attributes of endolymphatic hydrops, such as the origins of low-frequency hearing loss, a reliable model is needed. The guinea pig is a&#160;good model because it hears in the low-frequency regions that are putatively affected by endolymphatic hydrops. Previous research has demonstrated that endolymphatic hydrops can be induced surgically via intradural or extradural approaches that involve drilling on the endolymphatic duct and sac. However, whether it was possible to create an endolymphatic hydrops model using an extradural approach that avoided dangerous drilling on the endolymphatic duct and sac was unknown. The objective of this study was to demonstrate a revised extradural approach to induce experimental endolymphatic hydrops at 30 days post-operatively by obliterating the endolymphatic sac and injuring the endolymphatic duct with a fine pick. The sample size consisted of seven guinea pigs. Functional measurements of hearing were made and temporal bones were subsequently harvested for histologic analysis. The approach had a success rate of 86% in achieving endolymphatic hydrops. The risk of cerebrospinal fluid leak was minimal. No perioperative deaths or injuries to the posterior semicircular canal occurred in the sample. The presented method demonstrates a safe and reliable way to induce endolymphatic hydrops at a relatively quick time point of 30 days. The clinical implications are that the presented method provides a reliable model to further explore the origins of low-frequency hearing loss that can be associated endolymphatic hydrops.

This article demonstrates an extradural approach to obliterate the guinea pig endolymphatic sac and injure the endolymphatic duct with a fine pick in order to induce experimental endolymphatic hydrops.

Un enfoque quirúrgico revisado para inducir hidrops endoifáticos en el cerdo de Guinea

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) ...

Tecnología DREADDs y toma de muestras de sangre automatizadas

Remote Neuronal Activation Coupled with Automated Blood Sampling to Induce and Measure Circulating Luteinizing Hormone in Mice

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of numerous inputs and neuronal populations in the modulation of LH release is still unknown. Measuring changes in LH levels in mice is often a challenge since they are easily disrupted by environmental stress. Current techniques to measure LH release and pulsatility require long-term training for mice to adapt to manipulation stress, certain restraint, the presence of the investigator, and working on individual animals, reducing its usefulness for many research questions.
This paper presents a technique to remotely activate specific neuronal populations using Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) technology coupled with automated sequential blood sampling in conscious, freely moving, and undisturbed mice. We first describe the stereotaxic surgery protocol to deliver adeno-associated virus (AAV) vectors expressing DREADDs to specific neuronal populations. Next, we describe the protocol for carotid artery and jugular vein cannulation and postsurgical connection to the CULEX automated blood sampling system. Finally, we describe the protocol for clozapine-N-oxide intravenous injection for remote neuronal activation and automated blood collection. This technique allows for programmed automated sampling every 5 min or longer for a given period, coupled with intravenous substance injection at a desired time point or duration. Overall, we found this technique to be a powerful approach for research on neuroendocrine control.

Autor destacado: Infusión automatizada y muestreo de sangre para un análisis hormonal preciso en ratones conscientes

Luteinizing hormone (LH) pulsatility is a hallmark of reproductive function. We describe a protocol for remote activation of specific neuronal populations linked to serial automated blood collection. This technique allows timed hormonal modulation, multiplexing, and minimizing manipulation effects on LH levels in conscious freely moving, and undisturbed animals.

Activación neuronal remota junto con muestreo de sangre automatizado para inducir y medir la hormona luteinizante circulante en ratones

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of ...